黑皮冬瓜LINE逆轉座子RT序列的克隆與特征分析

趙芹等

摘 要 根據LINE逆轉座子逆轉錄酶保守序列設計簡并引物，PCR擴增黑皮冬瓜“B98K”基因組DNA，獲得580 bp左右目的條帶。將PCR產物回收、克隆并測序，獲得23條逆轉酶序列，利用生物信息學軟件分析其長度變異、堿基變化、相似性及系統進化關系。結果表明：這些序列長度在557～593 bp區間變異，同源比對核苷酸序列相似性為39.0%～99.3%，存在高度異質性，主要表現為缺失突變、移碼突變與終止密碼子突變，核苷酸序列聚類分析分為4個家族，家族1與家族2分別包含15和5個成員，占總序列數的65.22%和21.74%。對推導的氨基酸序列分析發現，第12位氨基酸殘基處存在一保守的苯丙氨酸（Phe），多處位置存在半保守氨基酸殘基；氨基酸序列相似性在20.0%～99.5%之間；其中8條序列可能具有轉錄活性，8條與15條序列分別發生移碼與終止密碼子突變。與已知物種構建系統發育進化樹，發現冬瓜LINE逆轉座子RT序列較保守，且與擬南芥、李、油菜等有較近的親緣關系。研究結果為后續利用分子標記研究冬瓜種質遺傳變異及基因組進化奠定基礎。

關鍵詞 黑皮冬瓜；LINE逆轉座子；逆轉錄酶；序列分析

中圖分類號 S642.3 文獻標識碼 A

Abstract Degenerate primers were designed according to the RT conserved motifs of LINE retrotransposons to amplify genomic DNA of black wax gourd“B98K”for investigating and understanding genetic relationship， characteristics and evolution of LINE retrotransposon families from black wax gourd. The PCR products of 580 bp were obtained and cloned to pMD18-T vector， and the positive clones were screened and sequenced. The full-length variation， base composition， homology and phylogenetic relationship of twenty-three different sequences were analyzed by bio-informatics softwares. The results indicated that length of RT sequences concentrated on the region of 557-593 bp， and sequence similarities varied from 39.0%-99.3%. These sequences showed high heterogeneity which was mainly characterized by deletion， frameshift and termination codon mutation. Four families were divided by alignment analyses of nucleotide sequences， and family1 and family2 contained most members of black wax gourd LINE retrotransposons and accounted for 65.22% and 21.74%， respectively. The analysis results of deduced amino acid sequences showed that there is a conservative phenylalanine（phe）in the 12th amino acid residue position of the translated sequences and lots of semi-conservative points appeared in many positions. Eight sequences were found to have transcriptional activity， moreover eight and fifteen sequences presented termination codon and frameshift mutations respectively， and amino acid sequence similarities ranged from 20.0% to 99.5%. The phylogenic tree constructed by these sequences and other RT sequences from GenBank showed that RT sequences of LINE retrotransposons in black wax gourd were conservative， and shared high homology with other species like Arabidopsis， plum and rape， etc. This work offers the basis for developing molecular markers to analyze the genetic variability and research the origin and evolution pathways of black wax gourd.

Key words Black wax gourd； LINE retrotransposons； Reverse transcriptase； Sequence analysis

doi 10.3969/j.issn.1000-2561.2015.03.004

冬瓜（Benincasa hispida Cogn.）為冬瓜屬一年生蔓性植物，原產中國與印度，現廣泛分布于亞洲的熱帶、亞熱帶和溫帶地區，在中國種植歷史悠久，南北地區分布廣泛[1]。冬瓜易栽培，產量高，耐貯運，保健藥用價值高[2-3]，是華南地區北運和出口創匯的重要蔬菜品種，對調節蔬菜淡季、保證蔬菜周年供應起著非常重要的作用[4]。冬瓜種質資源豐富，但目前對其種質資源分類評價研究較少，前人主要依據生態學性狀或RAPD與ISSR標記開展此類分析[5-7]，而不同種形成與進化之間的關系尚不清楚。……